PROJECT SUMMARY/ABSTRACT Women are exposed to sex hormones throughout their lifetimes, putting them at significantly higher risk for cardiovascular disease which is typically pathologic clotting (venous thrombosis/thromboembolism), and resulting in significant morbidity and mortality. Excessive exposure to estrogens due to sex and gender occurs with menstruation, endocrine disorders, and exogenous supplementation for contraception, hormone replacement, and gender transition. Although it is known that sex hormones induce expression of a number of coagulation factors, the pathways and mechanisms that connect estrogen to the coagulation system, as well as why there is progression to thrombosis, is poorly understood. Identification of these mediators are central to any comprehensive understanding of the underlying pathophysiology, could help ascertain patients at higher risk for thrombosis, and might also pinpoint future therapeutic targets. One of the reasons for the knowledge gap is the lack of an animal model that develops estrogen-induced thrombosis. We propose to exploit the powerful genetics of the zebrafish model system to identify the pathways that connect sex hormones to thromboembolism. This project will leverage highly innovative technologies, including genome editing nucleases, next generation sequencing, and small molecule analysis in the context of the zebrafish model. In preliminary studies, we have demonstrated widespread conservation of the coagulation system in zebrafish and sex hormone induction of thrombosis in embryos and larvae, including fluorescently-tagged and easily visible estrogen-induced fibrin thrombi. These studies will identify the critical proteins that mediate the link between sex hormones and coagulation factors. This will result in the rapid development of a candidate panel of factors that will enhance our understanding of sex hormone-induced thrombosis, and potentially allow stratification of at risk patients. Novel genes identified will also yield potential therapeutic targets and preventative strategies, and will inform future translational studies. Pathway analysis using small molecules will also identify potential therapeutic compounds that might treat or prevent estrogen-induced thrombosis. These studies will provide insights into the effects of sex and gender on disease, and are aligned with a number of the objectives in Strategic Goal 1 of the 2019-2023 Trans-NIH Strategic Plan for Women's Health Research, including investigation of conditions that specifically affect the cardiovascular, menstrual, and gynecologic health of women.